Murr, M. | bioRxiv Science

bioRxiv2026

Development of a multi-species luciferase-based double antigen ELISA for the detection of antibodies against Influenza A virus H5 clade 2.3.4.4b

The highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 represent a major threat to animal and public health. The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses and a high frequency of spillover events to unexpected novel mammalian hosts such as dairy cows. The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission. Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface. For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screenings are critically needed. The newly developed double antigen ELISA format employing a luminescence-based detection technology has demonstrated to comply with such prerequisites. The assay allowed the detection of H5-specific antibodies even early after infection or vaccination in a wide range of birds and mammals including humans. It further demonstrated superior analytical sensitivity and high specificity for antibodies directed against H5 hemagglutinin of clade 2.3.4.4b as no cross-reactivity with other hemagglutinin subtypes was observed. Thus, the assay represents a valuable contribution to existing serological diagnostic tests for a clade-optimized detection of influenza A virus antibodies in a broad range of species. ImportanceThe ongoing HPAIV H5N1 panzootic has caused numerous outbreaks in domestic and wild animals with frequent spillover events to unexpected host species, which underscores the importance of an intensified surveillance. However, sensitive and specific multi-species serological assays represent a major gap. For this purpose, we developed a novel double antigen ELISA which employs an innovative luminescence-based read-out strategy. The test allowed a highly sensitive and specific detection of H5-specific antibodies even early after infection or vaccination in a wide range of avian and mammalian species including humans. It therefore represents a significant contribution to improving species-independent serological diagnostic tools for the detection of influenza A virus antibodies.

microbiology↗

bioRxiv2023

Oral immunization of goats and foxes with a recombinant NDV vectored rabies vaccine

Vaccination of the reservoir species is a key component in the global fight against rabies. For wildlife reservoir species and hard to reach spillover species (e. g. ruminant farm animals), oral vaccination is the only solution. In search for a novel potent and safe oral rabies vaccine, we generated a recombinant vector virus based on lentogenic Newcastle disease virus (NDV) strain Clone 30 that expresses the glycoprotein G of rabies virus (RABV) vaccine strain SAD L16 (rNDV_GRABV). Transgene expression and virus replication was verified in avian and mammalian cells. To test immunogenicity and viral shedding, in a proof-of-concept study six goats and foxes, representing herbivore and carnivore species susceptible to rabies, each received a single dose of rNDV_GRABV (108.5 TCID50/animal) by direct oral application. For comparison, three animals received the similar dose of the empty viral vector (rNDV). All animals remained clinically inconspicuous during the trial. Viral RNA could be isolated from oral and nasal swabs until four (goats) or seven days (foxes) post vaccination, while infectious NDV could not be re-isolated. After four weeks, three out of six rNDV_GRABV vaccinated foxes developed RABV binding and virus neutralizing antibodies. Five out of six rNDV_GRABV vaccinated goats displayed RABV G specific antibodies either detected by ELISA or RFFIT. Additionally, NDV and RABV specific T cell activity was demonstrated in some of the vaccinated animals by detecting antigen specific interferon {gamma} secretion in lymphocytes isolated from pharyngeal lymph nodes. In conclusion, the NDV vectored rabies vaccine rNDV_GRABV was safe and immunogenic after a single oral application in goats and foxes, and highlight the potential of NDV as vector for oral vaccines in mammals. Author SummaryOral vaccination of rabies reservoir and spill-over species is the key to control the disease and prevent human rabies. In the past, baits containing live-attenuated rabies vaccines decreased significantly carnivore-mediated rabies in Central and Western Europe as well as North America. However, certain susceptible species are refractory to the oral immunization using so far licensed vaccines. Our vector vaccine based on avian Newcastle disease virus (NDV) has the potential to contribute to the targeted rabies eradication as it was safe and immunogenic after oral administration in goats and foxes. A single vaccine application elicited a rabies virus (RABV) specific systemic humoral immune response in the majority of the vaccinated animals as well as RABV specific T cells in some of the vaccinated animals. NDV can be manufactured at low-cost using already existing infrastructure of influenza vaccines, opening new possibilities especially for middle- and low-income countries that suffer under the economically burden of rabies.

microbiology↗